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Initial studies examining the effects of decreasing oxidative stress in several diseases, including cardiovascular disease, have used exogenous antioxidant supplements such as vitamins C and E. However, the protective effect of exogenous antioxidants has been disappointing and in some cases supplementation increased mortality [

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Nrf2 is constitutively expressed but is marked for ubiquitination by association with Kelch-like ECH-associated protein 1 (Keap1) in the cytosol. Activation of Nrf2 occurs when it is released from Keap1 and translocates to the nucleus. In the nucleus, Nrf2 heterodimerizes with small Maf or Jun proteins and binds to the antioxidant response element (ARE) in the promoter region of several hundred genes including many phase II antioxidant enzymes subsequently initiating transcription [

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The purpose of this study was to determine (1) if treatment with Protandim-induces Nrf2 nuclear translocation and phase II antioxidant enzyme protein expression in human coronary artery endothelial cells (HCAEC), (2) if treatment with Protandim protects HCAEC from apoptosis induced by an oxidant challenge, and (3) if Nrf2 mediates Protandim induced protection from an oxidative challenge. We hypothesized that Protandim treatment would induce Nrf2 nuclear localization and phase II antioxidant enzyme protein expression, and Protandim treatment prior to an oxidant challenge would afford cells protection in a Nrf2 dependent manner.

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Results

3.1. Antioxidant Enzyme Protein Expression Is Elevated in Response to Protandim TreatmentHCAEC were treated with Protandim concentrations of 0 to 50μg/mL in 5μg/mL increments to determine a profile of Nrf2 activation as measured by HO-1 protein content. HO-1 protein went from barely detectable in control conditions to 8–10-fold greater in cells treated with 20–30μg/mL (Figure 1(a)). Concentrations higher than 30μg/mL induced morphological changes. In all subsequent treatments 20μg/mL Protandim was used. HO-1 protein was visible after 1hr of Protandim treatment and became significant and sustained from 4hrs through the longest treatment period of 12hrs (data not shown). To confirm treatment concentration and duration on multiple antioxidant enzymes we determined that 20μg/mL Protandim for 12hrs induced HO-1 (778% of control + 82.25 P < 0.01), SOD1 (125.9% of control + 6.05 P < 0.01), NQO1 (126% of control + 6.5 P < 0.01), and GR (119.5% of control + 7.00 P < 0.05) (Figure 1(b)). All subsequent treatments used Protandim at a concentration of 20μg/mL for 12hrs unless otherwise noted. Figure 1Protandim treatment induces phase II antioxidant enzyme protein expression in HCAEC. (a) HCAECs were treated with 1–50μg/mL of Protandim for 12hrs prior to measuring HO-1 protein expression. The HO-1 signal was verified ...

3.2. Protandim Stimulates Nrf2 Nuclear LocalizationHCAECs were treated with Protandim for up to 12hrs and subsequently visualized using immunocytochemistry to determine Nrf2 nuclear localization. Nrf2 content and nuclear localization were elevated as soon as 1hr after Protandim treatment initiation. Induction remained with treatments of 2, 4, 8, and 12hrs (12hrs was the longest treatment duration examined) (Figure 2). Figure 2Protandim induced Nrf2 expression and nuclear localization. HCAECs were treated with Protandim for 1hr, following which immunofluorescence was used to visualize changes in Nrf2 expression and localization. Following Protandim treatment Nrf2 signal ...

3.3. Protandim Protects HCAEC from Oxidative Challenge Induced ApoptosisHCAECs were treated for 12hrs with Protandim or vehicle control followed by a 4hr exposure to 1.25μM HO and induction of apoptosis was measured by TUNEL assay. Protandim was removed prior to HO, exposure. In vehicle controls apoptosis was induced in 34% of cells (Figures 3(a) and 3(b)) while only 6% of cells treated with Protandim prior to HO were apoptotic (P < 0.01 compared to vehicle control) (Figures 3(a) and 3(b)). Figure 3Protandim treatment protects HCAEC against an oxidative stress. HCAEC were treated with Protandim for 12hrs, followed by HO 4hrs. (a) Treatment of HCAEC with Protandim prior to an oxidative challenge resulted in significantly fewer ...

3.4. Nrf2 Silencing Diminishes Protandim-Induced Increases in HO-1 and Protection from an Oxidative ChallengeNrf2 was silenced using siRNA prior to Protandim treatment to determine if Protandim induced increases in antioxidant enzyme expression and protection occur through Nrf2 activation. Nrf2 silencing prior to Protandim treatment significantly inhibited (P < 0.01) Protandim-induced HO-1 protein expression compared to both the no RNA condition, and control RNA condition. (Figures 4(a) and 4(b)). There were no differences between no RNA and control RNA conditions with or without Protandim. Figure 4Silencing of Nrf2 abrogated Protandim-induced increases in HO-1 expression. (a) Cells were treated with no RNA, control RNA, or Nrf2 siRNA prior to 12hrs Protandim, then HO-1 expression determined by Western blotting. (b) HO-1 expression in response ...Nrf2 was then silenced prior to Protandim treatment and an oxidative challenge. In cells receiving no RNA and cells that received control RNA, 30–40% of cells underwent apoptosis (Figures 5(a) and 5(b). Nrf2 siRNA treatment prior to HO resulted in apoptosis in 80% of cells (P < 0.0001) compared to no RNA conditions and control RNA (Figures 5(a) and 5(b)). In both no RNA and control RNA conditions, Protandim treatment prior to HO resulted in significantly fewer apoptotic cells (P < 0.0001) (Figure 5(b)). The number of apoptotic cells in the no RNA condition compared to the control RNA condition was not significantly different with or without Protandim (P = 0.413 no Protandim, P = 0.093 with Protandim). Protandim prior to Nrf2 siRNA also significantly protected cells from apoptosis (P = 0.023); however the amount of protection afforded by the Protandim in this condition was significantly less than in no RNA and control RNA conditions (P < 0.01 and P < 0.05, resp.) (Figure 5(c)). Figure 5Protandim treatment following Nrf2 silencing protected HCAEC against an oxidative challenge; however, protection following Nrf2 silencing was significantly diminished compared to controls. (a) Silencing of Nrf2 resulted in a significant increase in the ...

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